Cargando…

Differential equation methods for simulation of GFP kinetics in non–steady state experiments

Genetically encoded fluorescent proteins, combined with fluorescence microscopy, are widely used in cell biology to collect kinetic data on intracellular trafficking. Methods for extraction of quantitative information from these data are based on the mathematics of diffusion and tracer kinetics. Cur...

Descripción completa

Detalles Bibliográficos
Autor principal: Phair, Robert D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003217/
https://www.ncbi.nlm.nih.gov/pubmed/29367436
http://dx.doi.org/10.1091/mbc.E17-06-0396
_version_ 1783332334896939008
author Phair, Robert D.
author_facet Phair, Robert D.
author_sort Phair, Robert D.
collection PubMed
description Genetically encoded fluorescent proteins, combined with fluorescence microscopy, are widely used in cell biology to collect kinetic data on intracellular trafficking. Methods for extraction of quantitative information from these data are based on the mathematics of diffusion and tracer kinetics. Current methods, although useful and powerful, depend on the assumption that the cellular system being studied is in a steady state, that is, the assumption that all the molecular concentrations and fluxes are constant for the duration of the experiment. Here, we derive new tracer kinetic analytical methods for non–steady state biological systems by constructing mechanistic nonlinear differential equation models of the underlying cell biological processes and linking them to a separate set of differential equations governing the kinetics of the fluorescent tracer. Linking the two sets of equations is based on a new application of the fundamental tracer principle of indistinguishability and, unlike current methods, supports correct dependence of tracer kinetics on cellular dynamics. This approach thus provides a general mathematical framework for applications of GFP fluorescence microscopy (including photobleaching [FRAP, FLIP] and photoactivation to frequently encountered experimental protocols involving physiological or pharmacological perturbations (e.g., growth factors, neurotransmitters, acute knockouts, inhibitors, hormones, cytokines, and metabolites) that initiate mechanistically informative intracellular transients. When a new steady state is achieved, these methods automatically reduce to classical steady state tracer kinetic analysis.
format Online
Article
Text
id pubmed-6003217
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher The American Society for Cell Biology
record_format MEDLINE/PubMed
spelling pubmed-60032172018-06-20 Differential equation methods for simulation of GFP kinetics in non–steady state experiments Phair, Robert D. Mol Biol Cell Articles Genetically encoded fluorescent proteins, combined with fluorescence microscopy, are widely used in cell biology to collect kinetic data on intracellular trafficking. Methods for extraction of quantitative information from these data are based on the mathematics of diffusion and tracer kinetics. Current methods, although useful and powerful, depend on the assumption that the cellular system being studied is in a steady state, that is, the assumption that all the molecular concentrations and fluxes are constant for the duration of the experiment. Here, we derive new tracer kinetic analytical methods for non–steady state biological systems by constructing mechanistic nonlinear differential equation models of the underlying cell biological processes and linking them to a separate set of differential equations governing the kinetics of the fluorescent tracer. Linking the two sets of equations is based on a new application of the fundamental tracer principle of indistinguishability and, unlike current methods, supports correct dependence of tracer kinetics on cellular dynamics. This approach thus provides a general mathematical framework for applications of GFP fluorescence microscopy (including photobleaching [FRAP, FLIP] and photoactivation to frequently encountered experimental protocols involving physiological or pharmacological perturbations (e.g., growth factors, neurotransmitters, acute knockouts, inhibitors, hormones, cytokines, and metabolites) that initiate mechanistically informative intracellular transients. When a new steady state is achieved, these methods automatically reduce to classical steady state tracer kinetic analysis. The American Society for Cell Biology 2018-03-15 /pmc/articles/PMC6003217/ /pubmed/29367436 http://dx.doi.org/10.1091/mbc.E17-06-0396 Text en © 2018 Phair. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. http://creativecommons.org/licenses/by-nc-sa/3.0/ This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.
spellingShingle Articles
Phair, Robert D.
Differential equation methods for simulation of GFP kinetics in non–steady state experiments
title Differential equation methods for simulation of GFP kinetics in non–steady state experiments
title_full Differential equation methods for simulation of GFP kinetics in non–steady state experiments
title_fullStr Differential equation methods for simulation of GFP kinetics in non–steady state experiments
title_full_unstemmed Differential equation methods for simulation of GFP kinetics in non–steady state experiments
title_short Differential equation methods for simulation of GFP kinetics in non–steady state experiments
title_sort differential equation methods for simulation of gfp kinetics in non–steady state experiments
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003217/
https://www.ncbi.nlm.nih.gov/pubmed/29367436
http://dx.doi.org/10.1091/mbc.E17-06-0396
work_keys_str_mv AT phairrobertd differentialequationmethodsforsimulationofgfpkineticsinnonsteadystateexperiments